CHIMERIC PROTEINS COMPRISING BORRELIA POLYPEPTIDES: USES THEREFOR

Abstract: Novel chimeric nucleic acids, encoding chimeric Borrelia proteins consisting of at least two antigenic polypeptides from corresponding and/or non-corresponding proteins from the same and/or different species of Borrelia, are disclosed. Chimeric proteins encoded by the nucleic acid sequences are also disclosed. The chimeric proteins are useful as vaccine immunogens against Lyme borreliosis, as well as for immunodiagnostic reagents.

Description: CHIMERIC PROTEINS COMPRISING BORRELIA POLYPEPTIDES; USES THEREFOR Background of the Invention Lyme borreliosis is the most common tick-borne infectious disease in North America, Europe, and northern Asia. The causative bacterial agent of this disease, Borrelia burgdorferi , was first isolated and cultivated in 1982 (Burgdorferi, W.A. et al.. Science 216; 1317-1319 (1982); Steere, A.R. et al., N. Enσl. J. Med. 308; 733-740 (1983)). With that discovery, a wide array of clinical syndromes, described in both the European and American literature since the early 20th century, could be attributed to infection by B . burgdorferi (Afzelius, A., Acta Derm. Venereol. 2; 120- 125 (1921); Bannwarth, A., Arch. Psychiatr. Nervenkrankh. 117; 161-185 (1944) ; Garin, C. and A. Bujadouz, J. Med. Lyon 71; 765-767 (1922) ; Herxheimer, K. and K. Hartmann, Arch. Dermatol. Svphilol. 61; 57-76, 255-300 (1902)). The immune response to B . burgdorferi is characterized by an early, prominent, and persistent humoral response to the end of lagellar protein, p41 (fla) , and to a protein constituent of the protoplasmic cylinder, p93 (Szczepanski, A., and J.L. Benach, Microbiol. Rev. 55;21 (1991)). The p41 flagellin antigen is an immunodominant protein; however, it shares significant homology with flagellins of other microorganisms and therefore is highly cross reactive. The p93 antigen is the largest immunodominant antigen of B . burgdorferi . Both the p41 and p93 proteins are physically cryptic antigens, sheathed from the immune system by an outer membrane whose major protein constituents are the outer surface proteins A and B (OspA and OspB) . OspA is a basic lipoprotein of approximately 31 kd, which is encoded on a large linear plasmid along with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and J.L. Benach, Microbiol. Rev. 55;21 (1991)). Analysis of isolates of B . burgdorferi obtained from North America and Europe has demonstrated that OspA has antigenic variability, and that several distinct groups can be serologically and genotypically defined (Wilske, B. , et al . , World J. Microbiol. 7; 130 (1991)). Other Borrelia proteins demonstrate similar antigenic variability. Surprisingly, the immune response to these outer surface proteins tends to occur late in the disease, if at all (Craft, J. E. et al . , J. Clin Invest. 78: 934-939 (1986) ; Dattwyler, R.J. and B.J. Luft, Rheum. Clin. North Am. 15: 727-734 (1989)). Furthermore, patients acutely and chronically infected with B . burgdorferi respond variably to the different antigens, including OspA, OspB, OspC, OspD, p39, p41 and p93. Vaccines against Lyme borreliosis have been attempted. Mice immunized with a recombinant form of OspA are protected from challenge with the same strain of B . burgdorferi from which the protein was obtained (Fikrig, E. , et al . , Science 250: 553-556 (1990)). Furthermore, passively transferred anti-OspA monoclonal antibodies (Mabs) have been shown to be protective in mice, and vaccination with a recombinant protein induced protective immunity against subsequent infection with the homologous strain of B . burgdorferi (Simon, M.M. , et al . , J. Infect. Pis. 164: 123 (1991)). Unfortunately, immunization with a protein from one strain does not necessarily confer resistance to a heterologous strain (Fikrig, E. et al . , J. Immunol. 7: 2256-1160 (1992)), but rather, is limited to the homologous 'species' from which the protein was prepared. Furthermore, immunization with a single protein from a particular strain of Borrelia will not confer resistance to that strain in all individuals. There is considerable variation displayed in OspA and OspB, as well as p93, including the regions conferring antigenicity. Therefore, the degree and frequency of protection from vaccination with a protein from a single strain depend ' upon the response of the immune system to the particular variation, as well as the frequency of genetic variation in B. burgdorferi . Currently, a need exists for a vaccine which provides immunogenicity across species and to more epitopes within a species, as well as immunogenicity against more than one protein. Summary of the Invention The current invention pertains to chimeric Borrelia proteins which include two or more antigenic Borrelia polypeptides which do not occur naturally (in nature) in the same protein in Borrelia , as well as the nucleic acids encoding such chimeric proteins. The antigenic polypeptides incorporated in the chimeric proteins are derived from any Borrelia protein from any strain of Borrelia , and include outer surface protein (Osp) A, OspB, OspC, OspD, pl2, p39, p41, p66, and p93. The proteins from which the antigenic polypeptides are derived can be from the same strain of Borrelia , from different strains, or from combinations of proteins from the same and from different strains. If the proteins from which the antigenic polypeptides are derived are OspA or OspB, the antigenic polypeptides can be derived from either the portion of the OspA or OspB protein present between the amino terminus and the conserved tryptophan of the protein (referred to as a proximal portion) , or the portion of the OspA or OspB protein present between the conserved tryptophan of the protein and the carboxy terminus (referred to as a distal portion) . Particular chimeric proteins, and the nucleotide sequences encoding them, are set forth in Figures 23-37 and 43-46. The chimeric proteins of the current invention provide antigenic polypeptides of a variety of Borrelia strains and/or proteins within a single protein. Such" proteins are particularly useful in immunodiagostic assays to detect the presence of antibodies to native Borrelia in potentially infected individuals as well as to measure T-cell reactivity, and can therefore be used as immunodiagnostic reagents. The chimeric proteins of the current invention are additionally useful as vaccine immunogens against Borrelia infection. For a better understanding of the present invention together with other and further objects, reference is made to the following description, taken together with the accompanying drawings. Brief Description of the Drawings Figure 1 summarizes peptides and antigenic domains localized by proteolytic and chemical fragmentation of OspA. Figure 2 is a comparison of the antigenic domains depicted in Figure 1, for OspA in nine strains of B. burgdorferi . Figure 3 is a graph depicting a plot of weighted polymorphism versus amino acid position among 14 OspA variants. The marked peaks are: a) amino acids 132-145; b) amino acids 163-177; c) amino acids 208-221. The lower dotted line at polymorphism value 1.395 demarcates statistically significant excesses of polymorphism at p = 0.05. The upper dotted line at 1.520 is the same, except that the first 29 amino acids at the monomorphic N-terminus have been removed from the original analysis. Figure 4 depicts the amino acid alignment of residues 200 through 220 for OspAs from strains B31 and K48 as well as for the site-directed mutants 613, 625, 640, 613/625, and 613/640. Arrow indicates Trp216. Amino acid changes are underlined. Figure 5 is a helical wheel projection of residues 204-217 of B31 OspA. Capital letters indicate hydrophobic residues; lower case letters indicate hydrophilic residues; +/- indicate positively/negatively charged residues. Dashed line indicates division of the alpha-helix into hydrophobic arc (above the line) and polar arc (below the line) . Adapted from France et al . (Biochem. Biophvs. Acta 1120: 59 (1992)). Figure 6 depicts a phylogenic tree for strains of Borrelia described in Table I. The strains are as follows: 1 = B31; 2 = Pkal; 3 = ZS7; 4 = N40; 5 = 25015; 6 = K48; 7 - DK29; 8 = PHei; 9 = Ip90; 10 •= PTrob; 11 = ACAI; 12 = PGau; 13 = Ip3; 14 = PBo; 15 = PKo. Figure 7 depicts the nucleic acid sequence of OspA- B31 (SEW ID NO. 6) , and the encoded protein sequence (SEQ ID NO. 7) . Figure 8 depicts the nucleic acid sequence of OspA- K48 (SEQ ID NO. 8) , and the encoded protein sequence (SEQ ID NO. 9) . Figure 9 depicts the nucleic acid sequence of OspA- PGau (SEQ ID NO. 10) , and the encoded protein sequence (SEQ ID NO. 11) . Figure 10 depicts the nucleic acid sequence of OspA-25015 (SEQ ID NO. 12) , and the encoded protein sequence (SEQ ID NO. 13) . Figure 11 depicts the nucleic acid sequence of OspB-B31 (SEQ ID NO. 21) , and the encoded protein sequence (SEQ ID NO. 22) . Figure 12 depicts the nucleic acid sequence of 0spC-B31 (SEQ ID NO. 29) , and the encoded protein sequence (SEQ ID NO. 30) . Figure 13 depicts the nucleic acid sequence of OspC-K48 (SEQ ID NO. 31) , and the encoded protein sequence (SEQ ID NO. 32) . Figure 14 depicts the nucleic acid sequence of OspC-PKo (SEQ ID NO. 33) , and the encoded protein sequence (SEQ ID NO. 34) . Figure 15 depicts the nucleic acid sequence of OspC-pTrob (SEQ ID NO. 35) and the encoded protein sequence (SEQ ID NO. 36) . Figure 16 depicts the nucleic acid sequence of p93- B31 (SEQ ID NO. 65) and the encoded protein sequence (SEQ ID NO. 66) . Figure 17 depicts the nucleic acid sequence of p93- K48 (SEQ ID NO. 67) . Figure 18 depicts the nucleic acid sequence of p93- PBo (SEQ ID NO. 69) . Figure 19 depicts the nucleic acid sequence of p93- pTrob (SEQ ID NO. 71). Figure 20 depicts the nucleic acid sequence of p93- pGau (SEQ ID NO. 73) . Figure 21 depicts the nucleic acid sequence of p93- 25015 (SEQ ID NO. 75). Figure 22 depicts the nucleic acid sequence of p93- pKo (SEQ ID NO. 77) . Figure 23 depicts the nucleic acid sequence of the OspA-K48/OspA-PGau chimer (SEQ ID NO. 85) and the encoded chimeric protein sequence (SEQ ID NO. 86) . Figure 24 depicts the nucleic acid sequence of the 0spA-B31/0spA-PGau chimer (SEQ ID NO. 88) and the encoded chimeric protein sequence (SEQ ID NO. 89) . Figure 25 depicts the nucleic acid sequence of the 0spA-B31/0spA-K48 chimer (SEQ ID NO. 91) and the encoded chimeric protein sequence (SEQ ID NO. 92) . Figure 26 depicts the nucleic acid sequence of the OspA-B31/OspA-25015 chimer (SEQ ID NO. 94) and the encoded chimeric protein sequence (SEQ ID NO. 95) . Figure 27 depicts the nucleic acid sequence of the OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO. 97) and the encoded chimeric protein sequence (SEQ ID NO. 98) . Figure 28 depicts the nucleic acid sequence of the OspA-B31/OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO. 100) and the encoded chimeric protein sequence (SEQ ID NO. 101) . Figure 29 depicts the nucleic acid sequence of the OspA-B31/OspB-B31 chimer (SEQ ID NO. 103) and the encoded chimeric protein sequence (SEQ ID NO. 104) . Figure 30 depicts the nucleic acid sequence of the OspA-B31/OspB-B31/OspC-B31 chimer (SEQ ID NO. 106) and the encoded chimeric protein sequence (SEQ ID NO. 107) . Figure 31 depicts the nucleic acid sequence of the OspC-B31/OspA-B31/OspB-B31 chimer (SEQ ID NO. 109) and the encoded chimeric protein sequence (SEQ ID NO. 110) . Figure 32 depicts the nucleic acid sequence of the OspA-B31/p93-B31 chimer (SEQ ID NO. Ill) and the encoded chimeric protein sequence (SEQ ID NO. 112) . Figure 33 depicts the nucleic acid sequence of the OspB-B31/p41-B31 (122-234) chimer (SEQ ID NO. 113) and the encoded chimeric protein sequence (SEQ ID NO. 114) . Figure 34 depicts the nucleic acid sequence of the OspB-B31/p41-B31 (122-295) chimer (SEQ ID NO. 115) and the encoded chimeric protein sequence (SEQ ID NO. 116) . Figure 35 depicts the nucleic acid sequence of the OspB-B31/p41-B31 (140-234) chimer (SEQ ID NO. 117) and the encoded chimeric protein sequence (SEQ ID NO. 118) . Figure 36 depicts the nucleic acid sequence of the OspB-B31/p41-B31 (140-295) chimer (SEQ ID NO. 119) and the encoded chimeric protein sequence (SEQ ID NO. 120) . Figure 37 depicts the nucleic acid sequence of the OspB-B31/p41-B31 (122-234) /OspC-B31 chimer (SEQ ID NO. 121) and the encoded chimeric protein sequence (SEQ ID NO. 122) . Figure 38 depicts an alignment of the nucleic acid sequences for OspC-B31 (SEQ ID NO. 29) , OspC-PKo (SEQ ID NO. 33), OspC-pTrob (SEQ ID NO. 35), and OspC-K48 (SEQ ID NO. 31) . Nucleic acids which are identical to those in the lead nucleic acid sequence (here, OspC-B31) are represented by a period ( .) ; differing nucleic acids are shown in lower case letters. Figure 39 depicts an alignment of the nucleic acid sequences for OspD-pBO (SEQ ID NO. 123) , OspD-PGau (SEq ID NO. 124), OspD-DK29 (SEQ ID NO. 125), and OspD-K48 (SEQ ID NO. 126) . Nucleic acids which are identical to those in the lead nucleic acid sequence (here, OspD-pBo) are represented by a period (.); differing nucleic acids are shown in lower case letters. Figure 40 depicts the nucleic acid sequence of p41- B31 (SEq ID NO. 127) and then encoded protein sequence (SEQ ID NO. 128) . Figure 41 depicts an alignment of the nucleic acid sequences for p41-B31 (SEQ ID NO. 127) , p41-pKal (SEQ ID NO. 129), p41-PGau (SEQ ID NO. 51), p41-PBo (SEQ ID NO. 130), p41-DK29 (SEQ ID NO. 53), and p41-PKo (SEQ ID NO. 131) . Nucleic acids which are identical to those in the lead nucleic acid sequence (here, p41-B31) are represented by a period (.) ; differing nucleic acids are shown in lower case letters. Figure 42 depicts an alignment of the nucleic acid sequences for OspA-B31 (SEQ ID NO. 6) , OspA-pKal (SEQ ID NO. 132), OspA-N40 (SEQ ID NO. 133), OspA-ZS7 (SEQ ID NO. 134), OspA-25015 (SEQ ID NO. 12), OspA-pTrob (SEQ ID NO. 135) , OspA-K48 (SEQ ID NO. 8) , OspA-Hei (SEQ ID NO. 136), OspA-DK29 (SEQ ID NO. 49), OSpA-Ip90 (SEQ ID NO. 50), OspA-pBo (Seq ID NO. 55), OspA-Ip3 (SEQ ID NO. 56), OspA-PKo (SEQ ID NO. 57), OspA-ACAI (SEQ ID NO. 58), and OspA-PGau (SEQ ID NO. 10) . Nucleic acids which are identical to those in the lead nucleic acid sequence (here, OspA-B31) are represented by a period (.); differing nucleic acids are shown in lower case letters. Figure 43 depicts the nucleic acid sequence of the OspA-Tro/OspA-Bo chimer (SEQ ID NO. 137) and the encoded chimeric protein sequence (SEQ ID NO. 138) . Figure 44 depicts the nucleic acid sequence of the OspA-PGau/OspA-Bo chimer (SEQ ID NO. 139) and the encoded chimeric protein sequence (SEQ ID NO. 140) . Figure 45 depicts the nucleic acid sequence of the OspA-B31/OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO. 141) and the encoded chimeric protein sequence (SEQ ID NO. 142) . Figure 46 depicts the nucleic acid sequence of the OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO. 143) and the encoded chimeric protein sequence (SEQ ID NO. 144) . Detailed Description of the Invention The current invention pertains to chimeric proteins comprising antigenic Borrelia polypeptides which do not occur in nature in the same Borrelia protein. The chimeric proteins are a combination of two or more antigenic polypeptides derived from Borrelia proteins. The antigenic polypeptides can be derived from different proteins from the same species of Borrelia , or different proteins from different Borrelia species, as well as from corresponding proteins from different species. As used herein, the term "chimeric protein" describes a protein comprising two or more polypeptides which are derived from corresponding and/or non-corresponding native Borrelia protein. A polypeptide "derived from" a native Borrelia protein is a polypeptide which has an amino acid sequence the same as an amino acid sequence present in a Borrelia protein, an amino acid sequence equivalent to the amino acid sequence of a naturally occurring Borrelia protein, or an amino acid sequence substantially similar to the amino acid sequence of a naturally occurring Borrelia protein (e.g., differing by few amino acids) such as when a nucleic acid encoding a protein is subjected to site-directed mutagenesis. "Corresponding" proteins are equivalent proteins from different species or strains of Borrelia , such as outer surface protein A (OspA) from strain B31 and OspA from strain K48. The invention additionally pertains to nucleic acids encoding these chimeric proteins. As described below, Applicants have identified two separate antigenic domains of OspA and OspB which flank the sole conserved tryptophan present in OspA and in OspB. These domains share cross-reactivity with different genospecies of Borrelia . The precise amino acids responsible for antigenic variability were determined through site-directed mutagenesis, so that proteins with specific amino acid substitutions are available for the development of chimeric proteins. Furthermore, Applicants have identified immunologically important hypervariable domains in OspA proteins, as described below in Example 2. The first hypervariable domain of interest for chimeric proteins, Domain A, includes amino acid residues 120-140 of OspA, the second hypervariable domain, Domain B, includes residues 150- 180 and the third hypervariable domain, Domain C, includes residues 200-216 or 217 (depending on the position of the sole conserved tryptophan residue in the OspA of that particular species of Borrelia) (see Figure 3) . In addition, Applicants have sequenced the genes for several Borrelia proteins. These discoveries have aided in the development of novel recombinant Borrelia proteins which include two or more amino acid regions or sequences which do not occur in the same Borrelia protein in nature. The recombinant proteins comprise polypeptides from a variety of Borrelia proteins, including, but not limited to, OspA, OspB, OspC, OspD, pl2, p39, p41, p66, and p93. Antigenically relevant polypeptides from each of a number of proteins are combined into a single chimeric protein. In one embodiment of the current invention, chimers are now available which include antigenic polypeptides flanking a tryptophan residue. The antigenic polypeptides are derived from either the proximal portion from the tryptophan (the portion of the OspA or OspB protein present between the amino terminus and the conserved tryptophan of the protein) , or the distal portion from the tryptophan (the portion of the OspA or OspB protein present between the conserved tryptophan of the protein and the carboxy terminus) in OspA and/or OspB. The resultant chimers can be OspA-OspA chimers (i.e., chimers incorporating polypeptides derived from OspA from different strains of Borrelia) , OspA-OspB chimers, or OspB-OspB chimers, and are constructed such that amino acid residues amino-proximal to an invariant tryptophan are from one protein and residues carboxy- proximal to the invariant tryptophan are from the other protein. For example, one available chimer consists of a polypeptide derived from the amino-proximal region of OspA from strain B31, followed by the tryptophan residue, followed by a polypeptide derived from the carboxy-proximal region of OspA from strain K48 (SEQ ID NO. 92) . Another available chimer includes a polypeptide derived from the amino-proximal region of OspA from strain B31, and a polypeptide derived from the carboxy-proximal region of OspB from strain B31 (SEQ ID NO. 104) . If the polypeptide proximal to the tryptophan of these chimeric proteins is derived from OspA, the proximal polypeptide can be further subdivided into the three hypervariable domains (Domains A, B, and C) , each of which can be derived from OspA from a different strain of Borrelia . These chimeric proteins can further comprise antigenic polypeptides from another protein, in addition to the antigenic polypeptides flanking the tryptophan residue. In another embodiment of the current invention, chimeric proteins are available which incorporate antigenic domains of two or more Borrelia proteins, such as Osp proteins (Osp A, B, C and/or D) as well as pl2, p39, p41, p66, and/or p93. The chimers described herein can be produced so that they are highly soluble, hyper-produced in E. coli , and non-lipidated. In addition, the chimeric proteins can be designed to end in an affinity tag (His-tag) to facilitate purification. The recombinant proteins described herein have been constructed to maintain high levels of antigenicity. In addition, recombinant proteins specific for the various genospecies of Borrelia that cause Lyme disease are now available, because the genes from each of the major genospecies have been sequenced; the sequences are set forth below. These recombinant proteins with their novel biophysical and antigenic properties will be important diagnostic reagent and vaccine candidates. The chimeric proteins of the current invention are advantageous in that they retain specific reactivity to monoclonal and polyclonal antibodies against wild-type Borrelia proteins, are immunogenic, and inhibit the growth or induce lysis of Borrelia in vitro. Furthermore, in some embodiments, the proteins provide antigenic domains of two or more Borrelia strains and/or proteins within a single protein. Such proteins are particularly useful in immuno-diagostic assays. For example, proteins of the present invention can be used as reagents in assays to detect the presence of antibodies to native Borrelia in potentially infected individuals. These proteins can also be used as immunodiagnostic reagents, such as in dot blots, Western blots, enzyme linked immunosorbed assays, or agglutination assays. The chimeric proteins of the present invention can be produced by known techniques, such as by recombinant methodology, polymerase chain reaction, or mutagenesis. Furthermore, the proteins of the current invention are useful as vaccine immunogens against Borrelia infection. Because Borrelia has been shown to be clonal, a protein comprising antigenic polypeptides from a variety of Borrelia proteins and/or species, will provide immunoprotection for a considerable time when used in a vaccine. The lack of significant intragenic recombination, a process which might rapidly generate novel epitopes with changed antigenic properties, ensures that Borrelia can only change antigenic type by accumulating mutational change, which is slow when compared with recombination in generating different antigenic types. The chimeric protein can be combined with a physiologically acceptable carrier and administered to a vertebrate animal through standard methods (e.g., intravenously or intramuscularly, for example) . The current invention is illustrated by the following Examples, which are not to be construed to be limiting in any way. Example 1. Purification of Borrelia burαorferi Outer Surface Protein A and Analysis of Antibody Binding Domains This example details a method for the purification of large amounts of native outer surface protein A (OspA) to homogeneity, and describes mapping of the antigenic specificities of several anti-OspA MAbs. OspA was purified to homogeneity by exploiting its resistance to trypsin digestion. Intrinsic labeling with 14 C- palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino- terminal cysteine of the molecule. The reactivity of seven anti-OspA murine monoclonal antibodies to nine different Borrelia isolates was ascertained by Western blot analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains (see Figure 1). Domain 3, which included residues 190-220 of OspA, was reactive with protective antibodies known to agglutinate the organism in vitro , and included distinct specificities, some of which were not restricted to a genotype of B. Jurgrdorjferi. A. Purification of Native OspA Detergent solubilization of B. burgdorferi strips the outer surface proteins and yields partially-purified preparations containing both OspA and outer surface protein B (Osp B) (Barbour, A.G. et al . , Infect. Immun. 52 5^ : 549-554 (1986); Coleman, J.L. and J.L. Benach, J Infect. Pis. 155 (4): 756-765 (1987); Cunningham, T.M. ' et al . , Ann. NY Acad. Sci. 539; 376-378 (1988); Brandt, M.E. et al . , Infect. Immun. 58: 983-991 (1990); Sambri, V. and R. Cevenini, Microbiol. 14:307-314 (1991)). Although both OspA and OspB are sensitive to proteinase K digestion, in contrast to OspB, OspA is resistant to cleavage by trypsin (Dunn, J. et al . , Prot. Exp. Purif. 1: 159-168 (1990); Barbour, A.G. et al . , Infect. Immun. 5:94-100 (1984)). The relative insensitivity to trypsin is surprising in view of the fact that Osp A has a high (16% for B31) lysine content, and may relate to the relative configuration of Osp A and B in the outer membrane. Intrinsic Radiolabeling of Borrelia Labeling for lipoproteins was performed as described by Brandt et al . (Infect. Immun. 58:983-991 (1990)). 14 C-palmitic acid (ICN, Irvine, California) was added to the BSK II media to a final concentration of 0.5 μCi per illiliter (ml). Organisms were cultured at 34°C in this medium until a density of 10 8 cells per ml was achieved. Purification of OspA Protein from Borrelia Strain B31 Borrelia burgdorferi , either 14 C-palmitic acid- labeled or unlabeled, were harvested and washed as described (Brandt, M.E. et al . , Infect. Immun. 58:983- 991 (1990)). Whole organisms were trypsinized according to the protocol of Barbour et al . (Infect. Immun. 45:94- 100 (1984)) with some modifications. The pellet was suspended in phosphate buffered saline (PBS, lOmM, pH 7.2), containing 0.8% tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma, St. Louis, Missouri), the latter at a ratio of 1 μg per 10 8 cells. Reaction was carried out at 25°C for 1 hour, following ' which the cells were centrifuged. The pellet was washed in PBS with 100 μg/ml phenylmethylsulfonyl fluoride (PMSF) . Triton X-114 partitioning of the pellet was carried out as described by Brandt et al . (Infect. Immun. 58:983-991 (1990)). Following trypsin treatment, cells were resuspended in ice-cold 2% (v/v) Triton X-114 in PBS at 10 9 cells per ml. The suspension was rotated overnight at 4°C, and the insoluble fraction removed as a pellet after centrifugation at 10,000 X g for 15 minutes at 4°C. The supernatant (soluble fraction) was incubated at 37°C for 15 minutes and centrifuged at room temperature at 1000 X g for 15 minutes to separate the aqueous and detergent phases. The aqueous phase was decanted, and ice cold PBS added to the lower Triton phase, mixed, warmed to 37°C, and again centrifuged at 1000 X g for 15 minutes. Washing was repeated twice more. Finally, detergent was removed from the preparation using a spin column of Bio-beads SM2 (BioRad, Melville, New York) as described (Holloway, P.W., Anal. Biochem. 53:304-308 (1973)). Ion exchange chromatography was carried out as described by Dunn et al . (Prot. Exp. Purif. 1: 159-168 (1990)) with minor modifications. Crude OspA was dissolved in buffer A (1% Triton X-100, lOmM phosphate buffer (pH 5.0)) and loaded onto a SP Sepharose resin (Pharmacia, Piscataway, New Jersey) , pre-equilibrated with buffer A at 25°C. After washing the column with 10 bed-volumes of buffer A, the bound OspA was eluted with buffer B (1% Triton X-100, lOmM phosphate buffer (pH 8.0)). OspA fractions were detected by protein assay using the BCA method (Pierce, Rockford, Illinois) , or as radioactivity when intrinsically labeled material was fractionated. Triton X-100 was removed using a spin column of Bio-beads SM2. This method purifies OspA from an outer surface membrane preparation. In the absence of trypsin- treatment, OspA and B were the major components of the soluble fraction obtained after Triton partitioning of strain B31. In contrast, when Triton extraction was carried out after trypsin-treatment, the OspB band is not seen. Further purification of OspA-B31 on a SP Sepharose column resulted in a single band by SDS-PAGE. The yield following removal of detergent was approximately 2 mg per liter of culture. This method of purification of OspA, as described herein for strain B31, can be used for other isolates of Borrelia as well. For strains such as strain K48, which lack OspB, trypsin treatment can be omitted. Lipidation site of OspA-B31 14 C-palmitic acid labeled OspA from strain B31 was purified as described above and partially digested with endoproteinase Asp-N (data not shown) . Following digestion, a new band of lower molecular weight was apparent by SDS-PAGE, found by direct amino-terminal sequencing to begin at Asp^. This band had no trace of radioactivity by autoradiography (data not shown) . OspA and B contain a signal sequence (L-X-Y-C) similar to the consensus described for lipoproteins of E. coli , and it has been predicted that the lipidation site of OspA and B should be the amino-terminal cysteine (Brandt, M.E. et al . , Infect. Immun 58: 983-991 (1990)). The results presented herein support this prediction. B. Comparison of OspA Antibody Binding Regions in Nine Strains of Borrelia burgdorferi The availability of the amino acid sequenced for OspA from a number of different isolates, combined with peptide mapping and Western blot analysis, permitted the identification of the antigenic domains recognized by monoclonal antibodies (MAbs) and allowed inference of the key amino acid residues responsible for specific antibody reactivity. Strains of Borrelia burgdorferi Nine strains of Borrelia , including seven European strains and two North American strains, were used in this study of antibody binding domains of several proteins. Information concerning the strains is summarized in Table I, below. Table I. Representative Borrelia Strains ACA = patient w th acrodermatitis chronica atrophicans; EM = patient with erythema migrans; CSF = cerebrospinal fluid of patient with Lyme disease Strains K48, PGau and DK29 were supplied by R. Johnson, University of Minnesota; PKo and pTrob were provided by B. Wilske and V. Preac-Mursic of the Pettenkhofer Institute, Munich, Germany; and Ip3 and Ip90 were supplied by L. Mayer of the Center for Disease Control, Atlanta, Georgia. The North American strains included strain 25015, provided by J. Anderson of the Connecticut Department of Agriculture; and strain B31 (ATCC 35210) . Monoclonal Antibodies Seven monoclonal antibodies (MAbs) were utilized in this study. Five of the MAbs (12, 13, 15, 83 and 336) were produced from hybridomas cloned and subcloned as previously described (Schubach, W.H. , et al . , Infect. Immu . 59(6) :1911-1915 (1991)) . MA H5332 (Barbour, A.G. et al., Infect. Immun. 41:795-804 (1983)) was a gift from Drs . Alan Barbour, University of Texas, and MAb CIII.78 (Sears, J.E. et al . , J. Immunol. 147 (6) :1995-2000 (1991)) was a gift from Richard A. Flavell, Yale University. MAbs 12 and 15 were raised against whole sonicated B3; MAb 336 was produced against whole PGau; and MAbs 13 and 83 were raised to a truncated form of OspA cloned from the K48 strain and expressed in E. coli using the T7 RNA polymerase system (McGrath, B.C. et al . , Vaccines. Cold Spring Harbor Laboratory Press, Plainview, New York, pp. 365-370 (1993)) . All MAbs were typed as being Immunoglobulin G (IgG) . Methods of Protein Cleavage, Western Blotting, and Amino -Terminal Sequencing Prediction of the various cleavage sites was achieved by knowledge of the primary amino acid sequence derived from the full nucleotide sequences of OspA, many of which are currently available (see Table II, below) . Cleavage sites can also be predicted based on the peptide sequence of OspA, which can be determined by standard techniques after isolation and purification of OspA by the method described above. Cleavage of several OspA isolates was conducted to determine the localization of monoclonal antibody binding of the proteins. Hydroxylamine-HCl (HA) , N-chlorosuccinimide (NCS) , and cyanogen bromide cleavage of OspA followed the methods described by Bomstein (Biochem. 9 (12) :2408-2421 (1970)), Shechter et al . , (Biochem. 15 (23) :5071-5075 (1976)), and Gross (in Hirs, C.H.W. (ed) : Methods in Enzvmology. (N.Y. Acad. Press), ϋ:238-255 (1967)) respectively. Protease cleavage by endoproteinase, Asp-N (Boehringer Mannheim, Indianapolis, Indiana) , was performed as described by Cleveland D.W. et al . , (J. Biol. Chem. 252:1102-1106 (1977) ) . Ten micrograms of OspA were used for each reaction. The ratio of enzyme to OspA was approximately 1 to 10 (w/w) . Proteins and peptides generated by cleavage were separated by SDS-polyacrylamide gel electrophoresis (SDS- PAGE) (Laemmli, U.K., Nature (London) 227.:680-685 (1970)), and electroblotted onto immobilon Polyvinylidine Difluoride (PVDF) membranes (Ploskal, M.G. et al . , Biotechniσues 4 . :272-283 (1986)) . They were detected by a ido black staining or by immunostaining with murine MAbs, followed by alkaline phosphatase-conjugated goat antimouse IgG. Specific binding was detected using a 5-bromo-4-chloro-3- indolylphosphate (BCIP) /nitroblue tetrazolium (NBT) developer system (KPL Inc., Gathersburg, Maryland) . In addition, amino-terminal amino acid sequence analysis was carried out on several cleavage products, as described by Luft et al . (Infect. Immun. 57:3637-3645 (1989)) . Amido black stained bands were excised from PVDF blots and sequenced by Edman degradation using a Biosystems model 475A sequenator with model 120A PTH analyzer and model 900A control/data analyzer. Cleavage Products of Outer Surface Protein A Isolates Purified OspA-B31, labeled with 14 C-palmitic acid, was fragmented with hydroxylamine-HCl (HA) into two peptides, designated HA1 and HA2 (data not shown) . The HA1 band migrated at 27 KD and retained its radioactivity, indicating that the peptide included the lipidation site at the N-terminus of the molecule (data not shown) . From the predicted cleavage point, HA1 should correspond to residues 1 to 251 of 0spA-B31. HA2 had a MW of 21.6 KD by SDS-PAGE, with amino-terminal sequence analysis showing it to begin at Gly72, i.e. residues 72 to 273 of 0spA-B31. By contrast, HA cleaved OspA-K48 into three peptides, designated HA1, HA2, and HA3 with apparent MWs of 22KD, 16 KD and 12 KD, respectively. Amino-terminal sequencing showed HA1 to start at Gly72, and HA3 at Glyl42. HA2 was found to have a blocked amino-terminus, as was observed for the full-length OspA protein. HA1, 2 and 3 of OspA-K48 were predicted to be residues 72-274, 1 to 141 and 142 to 274, respectively. N-Chlorosuccinimide (NCS) cleaves tryptophan (W) , which is at residue 216 of OspA-B31 or residue 217 of OspA- K48 (data not shown) . NCS cleaved OspA-B31 into 2 fragments, NCS1, with MW of 23 KD, residues 1-216 of the protein, and NCS2 with a MW of 6.2 KD, residues 217 to 273 (data not shown) . Similarly, K 8 OspA was divided into 2 pieces, NCS1 residues 1-217, and NCS2 residues 218 to 274 (data not shown) . Cleavage of OspA by cyanogen bromide (CNBr) occurs at the carboxy side of methionine, residue 39. The major fragment, CNBrl, has a MW of 25.7 KD, residues 39-274 by amino-terminal amino acid sequence analysis (data not shown) . CNBr2 (about 4 KD) could not be visualized by amido black staining; instead, lightly stained bands of about 20 KD MW were seen. These bands reacted with anti- OspA MAbs, and most likely were degradation products due to cleavage by formic acid. Determination of Antibody Binding Domains for Anti - OspA Monoclonal Antibodies The cleavage products of 0spA-B31 and OspA-K48 were analyzed by Western blot to assess their ability to bind to the six different MAbs. Preliminary Western blot analysis of the cleavage products demonstrated that strains K48 and DK29 have similar patterns of reactivity, as do IP3, PGau and PKo. The OspA of strain PTrob was immunologically distinct from the others, being recognized only by MAb 336. MAb 12 recognized only the two North American strains, B31 and 25015. When the isolates were separated into genogroups, it was remarkable that all the MAbs, except MAb 12, crossed over to react with multiple genogroups. MAbl2, specific for 0spA-B31, bound to both HA1 and HA2 of 0spA-B31. However, cleavage of 0spA-B31 by NCS at residue Trp216 created fragments which did not react with MAbl2, suggesting that the relevant domain is near or is structurally dependent upon the integrity of this residue (data not shown) . MAb 13 bound only to OspA-K48, and to peptides containing the amino-terminus of that molecule (e.g. HA2; NCS1) . It did not bind to CNBrl residues 39 to 274. Thus the domain recognized by MAbl3 is in the amino- terminal end of OspA-K48, near Met38. MAbl5 reacts with the OspA of both the B31 and K48 . strains, and to peptides containing the N-terminus of OspA, such as HA1 of OspA-B31 and NCS1, but not to peptides HA2 of 0spA-B31 and HA1 of OspA-K48 (data not shown) . Both peptides include residue 72 to the C-terminus of the molecules. MAbl5 bound to CNBrl of OspA-K48, indicating the domain for this antibody to be residues 39 to 72, specifically near Gly72 (data not shown) . MAb83 binds to OspA-K48, and to peptides containing the C-terminal portion of the molecule, such as HA1. They do not bind to HA2 of OspA-K48, most likely because the C- terminus of HA2 of OspA-K48 ends at 141. Similar to MAbl2 and 0spA-B31, binding of MAbs 83 and CIII.78 is eliminated by cleavage of OspA at the tryptophan residue. Thus binding of MAbs 12, 83 and CIII.78 to OspA depends on the structural integrity of the Trp 216 residue, which appears to be critical for antigenicity. Also apparent is that, although these MAbs bind to a common antigenic domain, the precise epitopes which they recognize are distinct from one another given the varying degrees of cross-reactivity to these MAbs among strains. Although there is similar loss of binding activity of MAb336 with cleavage at Trp 216 , this MAb does not bind to HA1 of OspA-B31, suggesting the domain for this antibody includes the carboxy-terminal end of the molecule, inclusive of residues 251 to 273. Low MW peptides, such as HA3 (10 KD) and NCS2 (6KD) , of OspA-K48 do not bind this MAb on Western blots. In order to confirm this observation, we tested binding of the 6 MAbs with a recombinant fusion construct p3A/EC that contains a trpE leader protein fused with residues 217 to 273 of 0spA-B31 (Schubach, W.H. et al . , Infect. Immun. 59(6) : 1911-1915 (1991) ) . Only MAb336 reacted with this construct (data not shown) . Peptides and antigenic domains localized by fragmentation of OspA are summarized in Figure 1. Mapping of Domains to Define the Molecular Basis for the Serotype Analysis To define the molecular basis for the serotype analysis of OspA, we compared the derived amino acid sequences of OspA for the nine isolates (Figure 2) . At the amino terminus of the protein, these predictions can be more precise given the relatively small number of amino acid substitutions in this region compared to the carboxy terminus. Domain 1. which is recognized by MAbl3, includes residues Leu34 to Leu41. MAbl3 only binds to the OspA of species K48, DK29 and IP90. Within this region, residue 37 is variable, however Gly37 is conserved amongst the three reactive strains. When Gly37 is changed to Glu37, as it is in OspA of strains B31, pTrob, PGau, and PKo, MAbl3 does not recognize the protein (data not shown) . By similar analysis, it can be seen that Asp70 is a crucial residue for Domain 2, which includes residues 65 to 75 and is recognized by MAbl5. Domain 3 is reactive with MAbs H5332, 12 and 83, and includes residues 190-220. It is clear that significant heterogeneity exists between MAbs reactive with this domain, and that more than one conformational epitope must be contained within the sequence. Domain 4 binds MAb336, and includes residues 250 to 270. In this region, residue 266 is variable and therefore may be an important determinant. It is apparent, however, that other determinants of the reactivity of this monoclonal antibody reside in the region comprising amino acids 217-250. Furthermore, the structural integrity of Trp216 is essential for antibody reactivity in the intact protein. Finally, it is important to stress that Figure 2 indicates only the locations of the domains, and does not necessarily encompass the entire domain. Exact epitopes are being analyzed by site-directed mutagenesis of specific residues. Overall, evidence suggests that the N-terminal portion is not the immunodominant domain of OspA, possibly by virtue of its lipidation, and the putative function of the lipid moiety in anchoring the protein to the outer envelope. The C-terminal end is immunodominant and includes domains that account in part for structural heterogeneity (Wilske, B. et al., Med. Microbiol. Immunol. 181: 191-207 (1992)), and may provide epitopes for antibody neutralization (Sears, J.E. et al . , J . Immunol. 147 (6) : 1995-2000 (1991)), and relate to other activities, such as the induction of T-cell proliferation (Shanafel, M.M. , et al., J. Immunol. 148: 218-224 (1992)) . There are common epitopes in the carboxy-end of the protein that are shared among genospecies which may have immunoprotective potential (Wilske, B., et al . , Med. Microbiol. Immunol. 181: 191-207 (1992)) . Prediction of secondary structure on the basis of hydropathy analysis and circular dichroism and fluorescence spectroscopy measurements (McGrath, B.C., et al., Vaccines. Cold Spring Harbor Laboratory Press, Plainview, New York; pp. 365-370 (1993)) suggest domains 3 and 4 to be in a region of the molecule with a propensity to form alpha- helix, whereas domains 1 and 2 occur in regions predicted to be beta-sheets (see Figure 1) . These differences may distinguish domains in accessibility to antibody or to reactive T-cells (Shanafel, M.M. et al . , J. Immunol. 148: 218-224 (1992)) . Site-directed mutagenesis of specific epitopes, as described below in Example 2, aids in identifying exact epitopes. Example 2. Identification of an Immunologicallv Important Hypervariable Domain of the Major Outer Surface Protein A of Borrelia This Example describes epitope mapping studies using chemically cleaved OspA and TrpE-OspA fusion proteins. The studies indicate a hypervariable region surrounding the single conserved tryptophan residue of OspA (at residue 216, or in some cases 217), as determined by a moving window population analysis of OspA from fifteen European and North American isolates of Borrelia . The hypervariable region is important for immune recognition. Site-directed mutagenesis was also conducted to examine the hypervariable regions more closely. Fluorescence and circular dichroism spectroscopy have indicated that the conserved tryptophan is part of an alpha-helical region in which the tryptophan is buried in a hydrophobic environment (McGrath, B.C., et al., Vaccines, . Cold Spring Harbor Laboratory Press, Plainview, New York; pp. 365-370 (1993)) . More polar amino acid side-chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent. The hypervariability of these solvent-exposed residues among the various strains of Borrelia suggested that these amino acid residues may contribute to the antigenic variation in OspA. Therefore, site-directed mutagenesis was performed to replace some of the potentially exposed amino acid side chains in the protein from one strain with the analogous residues of a second strain. The altered proteins were then analyzed by Western Blot using monoclonal antibodies which bind OspA on the surface of the intact, non-mutated spirochete. The results indicated that certain specific amino acid changes near the tryptophan can abolish reactivity of OspA to these monoclonal antibodies. A. Verification of Clustered Polymorphisms in Outer Surface Protein A Sequences Cloning and sequencing of the OspA protein from fifteen European and North American isolates (described above in Table I) demonstrated that amino acid polymorphism is not randomly distributed throughout the protein; rather, polymorphism tended to be clustered in three regions of OspA. The analysis was carried out by plotting the moving, weighted average polymorphism of a window (a fixed length subsection of the total sequence) as it is slid along the sequence. The window size in this analysis was thirteen amino acids, based upon the determination of the largest number of significantly deviating points as established by the method of Tajima (J. Mol. Evol. 33: 470-473 (1991)) . The average weighted polymorphism was calculated by summing the number of variant alleles for each site. Polymorphism calculations were weighted by the severity of amino acid replacement (Dayhoff, M.O. et al., in: Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure NBRF. Washington, Vol. 5, Suppl. 3 : 345 (1978)) . The sum was normalized by the window size and plotted. The amino acid sequence position corresponds to a window that encompasses amino acids 1 through 13. Bootstrap resampling was used to generate 95% confidence intervals on the sliding window analysis. Since Borrelia has been shown to be clonal, the bootstrap analysis should give a reliable estimate of the expected variance out of polymorphism calculations. The bootstrap was iterated five hundred times at each position, and the mean was calculated from the sum of all positions. The clonal nature of Borrelia ensures that the stochastic variance that results from differing genealogical histories of the sequence positions (as would be expected if recombination were prevalent) will be minimized. This test verified that the three regions around the observed peaks all have significant excesses of polymorphism. Excesses of polymorphism were observed in the regions including amino acid residues 132-145, residues 163-177, and residues 208-221 (Figure 3) . An amino acid alignment between residues 200 and 220 for B31, K48 and the four site-directed mutants is shown in Figure 4. The amino acid 208-221 region includes the region of OspA which has been modeled as an oriented alpha-helix in which the single tryptophan residue at amino acid 216 is buried in a hydrophobic pocket, thereby exposing more polar amino acids to the solvent (Figure 5) (France, L.L., et al . , Biochem. Biophvs. Acta 1120: 59 (1992)) . These potentially solvent- exposed residues showed considerable variability among the OspAs from various strains and may be an important component of OspA antigenic variation. For the purposes of generating chimeric proteins, the hypervariable domains of interest are Domain A, which includes amino acid residues 120-140 of OspA; Domain B. which includes residues 150-180; and Domain C. which includes residues 200-216 or 217. B. Site-Directed Mutagenesis of the Hypervariable Region Site-directed mutagenesis was performed to convert residues within the 204-219 domain of the recombinant B31 OspA to the analogous residues of a European OspA variant, K48. In the region of OspA between residues 204 and 219, which includes the helical domain (amino acids 204-217) , there are seven amino acid differences between OspA-B31 and OspA-K48. Three oligonucleotides were generated, each containing nucleotide changes which would incorporate K48 amino acids at their analogous positions in the B31 OspA protein. The oligos used to create the site-directed mutants were: 5'-CTTAATGACTCTGACACTAGTGC-3' (#613, which converts threonine at position 204 to serine, and serine at 206 to threonine (Thr204-Ser, Thr206-Ser) ) (SEQ ID NO. 1) ; 5' -GCTACTAAAAAAACCGGGAAATGGAATTCA-3' (#625, which converts alanine at 214 to glycine, and alanine at 215 to lysine (Ala214-Gly, Ala215-Lys) ) (SEQ ID NO. 2) ; and 5'-GCAGCTTGGGATTCAAAAACATCCACTTTAACA-3' (#640, which converts asparagine at 217 to aspartate, and glycine at 219 to lysine (Asn217-Asp, Gly219-Lys) ) (SEQ ID NO. 3) . Site-directed mutagenesis was carried out by performing mutagenesis with pairs of the above oligos. Three site-directed mutants were created, each with two changes: OspA 613 (Thr204-Ser, Thr206-Ser) , OspA 625 (Ala214-Gly, Ala215-Lys) , and 640 (Asn217-Asp, Gly2l9-Lys) . There were also two proteins with four changes: OspA 613/625 (Thr204-Ser, Thr206-Ser, Ala214-Gly, Ala215-Lys) and OspA 613/640 (Thr204-Ser, Thr206-Ser, Asn217-Asp, Gly219-Lys) . Specifici ty of Antibody Binding to Epi topes of the Non-mutated Hypervariable Region Monoclonal antibodies that agglutinate spirochetes, including several which are neutralizing in vi tro, recognize epitopes that map to the hypervariable region around Trp216 (Barbour, A.G. et al . , Infect, and Immun. 41: 759 (1983) ; Schubach, W.H. et al . , Infect, and Immun. 59: 1911 (1991) ) . Western Blot analysis demonstrated that chemical cleavage of OspA from the B31 strain at Trp 216 abolishes reactivity of the protein with the agglutinating Mab 105, a monoclonal raised against B31 spirochetes (data not shown) . The reagent, n-chlorosuccinimide (NCS) , cleaves OspA at the Trp 216, forming a 23.2kd fragment and a 6.2kd peptide which is not retained on the Imobilon-P membrane after transfer. The uncleaved material binds Mab 105; however, the 23.2kd fragment is unreactive. Similar Western blots with a TrpE-OspA fusion protein containing the carboxy-terminal portion of the OspA protein demonstrated that the small 6.2kd piece also fails to bind Mab 105 (Schubach, W.H. et al . , Infect, and Immun. 59: 1911 (1991) ) . Monoclonal antibodies H5332 and H3TS (Barbour, A.G. et al . , Infect, and Immun. 41: 759 (1983)) have been shown by immunofluorescence to decorate the surface of fixed spirochetes (Wilske, B. et al . , World J. Microbiol. 7: 130 (1991) ) . These monoclonals also inhibit the growth of the organism in culture. Epitope mapping with fusion proteins has confirmed that the epitopes which bind these Mabs are conformationally determined and reside in the carboxy half of the protein. Mab H5332 is cross-reactive among all of the known phylogenetic groups, whereas Mab H3TS and Mab 105 seem to be specific to the B31 strain to which they were raised. Like Mab 105, the reactivities of H5332 and H3TS to OspA are abrogated by fragmentation of the protein at Trp216 (data not shown) . Mab 336 was raised to whole spirochetes of the strain P/Gau. It cross-reacts to OspA from group 1 (the group to which B31 belongs) but not to group 2 (of which K48 is a member) . Previous studies using fusion proteins and chemical cleavage have indicated that this antibody recognizes a domain of OspA in the region between residues 217 and 273 (data not shown) . All of these Mabs will agglutinate the B31 spirochete. Western Blot Analysis of Antibody Binding to Mutated Hypervariable Regions Mabs were used for Western Blot analysis of the site- directed OspA mutants induced in E. coli using the T7 expression system (Dunn, J.J. et al., Protein Expression and Purification 1: 159 (1990)) . E. coli cells carrying Pet9c plasmids having a site-directed OspA mutant insert were induced at mid-log phase growth with IPTG for four hours at 37°C. Cell lysates were made by boiling an aliquot of the induced cultures in SDS gell loading dye, and this material was then loaded onto a 12% SDS gell (BioRad mini-Protean II) , and electrophoresed. The proteins were then transferred to Imobilon-P membranes (Millipore) 70V, 2 hour at 4°C using the BioRad mini transfer system. Western analysis was carried out as described by Schubach et al . (Infect. Immun. 59: 1911 (1991) ) . Western Blot analysis indicated that only the 625 mutant (Ala214-Gly and Ala215-Lys) retained binding to the agglutinating monoclonal H3TS (data not shown) . However, the 613/625 mutant which has additional alterations to the amino terminus of Trp216 (Ser204-Thr and Thr206-Ser) did not bind this monoclonal. Both 640 and 613/640 OspAs which have the Asn217-Asp and Gly219-Lys changes on the carboxy- terminal side of Trp216 also failed to bind Mab H3TS. This indicated that the epitope of the B31 OspA which binds H3TS is comprised of amino acid side-chains on both sides of Trp216. The 613/625 mutant failed to bind Mabs 105 and H5332, while the other mutants retained their ability to bind these Mabs. This is important in light of the data using fusion proteins that indicate that Mab 105 behaves more like Mab H3TS in terms of its serotype specificity and binding to OspA (Wilske, B. et al . , Med. Microbiol. Immunol. 181: 191 (1992)) . The 613/625 protein has, in addition to the differences at residues Thr204 and Ser206, changes immediately amino-terminal to Trp216 (Ala214-Gly and Ala215-Lys) . The abrogation of reactivity of Mabs 105 and H5332 to this protein indicated that the epitopes of OspA which bind these monoclonals are comprised of residues on the amino-terminal side of Trp216. The two proteins carrying the Asn217-Asp and Gly219- Lys replacements on the carboxy-terminal side of Trp216 (OspAs 640 and 613/640) retained binding to Mabs 105 and H5332; however, they failed to react with Mab 336, a monoclonal which has been mapped with TrpE-OspA fusion proteins and by chemical cleavage to a more carboxy- terminal domain. This result may explain why Mab 336 failed to recognize the K48-type of OspA (Group 2) . It is clear that amino acids Ser204 and Thr206 play an important part in the agglutinating epitopes in the region of the B31 OspA flanking Trp216. Replacement of these two residues altered the epitopes of OspA that bind Mabs 105, H3TS and H5332. The ability of the 640 changes alone to abolish reactivity of Mab 336 indicated that Thr204 and Ser206 are not involved in direct interaction with Mab 336. The results indicated that the epitopes of OspA which are available to Mabs that agglutinate spirochetes are comprised at least in part by amino acids in the immediate vicinity of Trp216. Since recent circular dichroism analysis indicated that the structures of B31 and K48 OspA differ very little within this domain, it is unlikely that the changes made by mutation have radically altered the overall structure of the OspA protein (France, L.L. et al., Biochem. Biophys. Acta 1120: 59 (1992); and France et al . , Biochem. Biophvs Acta. submitted (1993)) . This hypothesis is supported by the finding that the recombinant, mutant OspAs exhibit the same high solubility and purification properties as the parent B31 protein (data not shown) . In summary, amino acid side-chains at Ser204 and Thr206 are important for many of the agglutinating epitopes. However, a limited set of conservative changes at these sites were not sufficient to abolish binding of all of the agglutinating Mabs. These results suggested that the agglutinating epitopes of OspA are distinct, yet may have some overlap. The results also supported the hypothesis that the surface-exposed epitope around Trp216 which is thought to be important for immune recognition and neutralization is a conformationally-determined and complex domain of OspA. EXAMPLE 3. Borrelia Strains and Proteins Proteins and genes from any strain of Borrelia can be utilized in the current invention. Representative strains are summarized in Table I, above. A. Genes Encoding Borrelia Proteins The chimeric peptides of the current invention can comprise peptides derived from any Borrelia proteins. Representative proteins include OspA, OspB, OspC, OspD, pl2, p39, p41 (fla) , p66, and p93. Nucleic acid sequences encoding several Borrelia proteins are presently available (see Table II, below) ; alternatively, nucleic acid sequences encoding Borrelia proteins can be isolated and characterized using methods such as those described below. Table II. References for Nucleic Acid Sequences for Several Proteins of Various Borrelia Strains Numbers with an "X" pre ix are GenBank data base accession numbers. SID = SEQ ID NO. B. Isolation of Borrelia Genes Nucleic acid sequences encoding full length, lipidated proteins from known Borrelia strains were isolated using the polymerase chain reaction (PCR) as described below. In addition, nucleic acid sequences were generated which encoded truncated proteins (proteins in which the lipidation signal has been removed, such as by eliminating the nucleic acid sequence encoding the first 18 amino acids, resulting in non-lipidated proteins) . Other proteins were generated which encoded polypeptides of a particular gene (i.e., encoding a segment of the protein which has a different number of amino acids than the protein does in nature) . Using similar methods as those described below, primers can be generated from known nucleic acid sequences encoding Borrelia proteins and used to isolate other genes encoding Borrelia proteins. Primers can be designed to amplify all of a gene, as well as to amplify a nucleic acid sequence encoding truncated protein sequences, such as described below for OspC, or nucleic acid sequences encoding a polypeptide derived from a Borrelia protein. Primers can also be designed to incorporate unique restriction enzyme cleavage sites into the amplified nucleic acid sequences. Sequence analysis of the amplified nucleic acid sequences can then be performed using standard techniques. Cloning and Sequencing of OspA Genes and Relevant Nucleic Acid Sequences Borrelia OspA sequences were isolated in the following manner: 100 μl reaction mixtures containing 50 mM KC1, 10 mM TRIS-HC1 (pH 8,3), 1.5 mM MgCl 2 , 200 μM each NTP, 2.5 units of TaqI DNA polymerase (Amplitaq, Perkin-Elmer/Cetus) and 100 pmol each of the 5' and 3' primers (described below) were used. Amplification was performed in a Perkin- Elmer/Cetus thermal cycler as described (Schubach, W.H. et al . , Infect. Immun. 59:1811-1915 (1991)) . The a plicon was visualized on an agarose gel by ethidium bromide staining. Twenty nanograms of the chloroform-extracted PCR product were cloned directly into the PC-TA vector (Invitrogen) by following the manufacturer's instructions. Recombinant colonies containing the amplified fragment were selected, the plasmids were prepared, and the nucleic acid sequence of each OspA was determined by the dideoxy chain- termination technique using the Sequenase kit (United States Biochemical) . Directed sequencing was performed with M13 primers followed by OspA-specific primers derived from sequences, previously obtained with M13 primers. Because the 5' and 3' ends of the OspA gene are highly conserved (Fikrig, E.S. et al . , J . Immunol. 7:2256-2260 (1992); Bergstrom, S. et al . , Mol. Microbiol. 3 : 479-486 (1989); Zumstein, G. et al . , Med. Microbiol. Immunol. 181: 57-70 (1992)), the 5' and 3' primers for cloning can be based upon any known OspA sequences. For example, the following primers based upon the OspA nucleic acid sequence from strain B31 were used: 5' -GGAGAATATATTATGAAA-3' (-12 to +6) (SEQ ID NO. 4) ; and 5' -CTCCTTATTTTAAAGCG-3' (+826 to +809) (SEQ ID NO. 5) . (Schubach, W.H. et al . , Infect. Immun 59:1811-1915 (1991)) . OspA genes isolated in this manner include those for strains B31, K48, PGau, and 25015; the nucleic acid sequences are depicted in the sequence listing as SEQ ID NO. 6 (OspA-B31) , SEQ ID NO. 8 (OspA-K48) , SEQ ID NO. 10 (OspA-PGau) , and SEQ ID NO. 12 (OspA-25015) . An alignment of these and other OspA nucleic acid sequences is shown in Figure 42. The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as ■ SEQ ID NO. 7 (OspA-B31) , SEQ ID NO. 9 (OspA-K48) , SEQ ID NO. 11 (OspA-PGau) , and SEQ ID NO. 13 (OspA-25015) . The following primers were used to generate specific nucleic acid sequences of the OspA gene, to be used to generate chimeric nucleic acid sequences (as described in Example 4) : 5'-GTCTGCAAAAACCATGACAAG-3' (plus strand primer #369) (SEQ ID NO. 14) ; 5' -GTCATCAACAGAAGAAAAATTC-3' (plus strand primer #357) (SEQ ID NO 15) ; 5' -CCGGATCCATATGAAAAAATATTTATTGGG-3' (plus strand primer #607) (SEQ ID NO. 16) ; 5'-CCGGGATCCATATGGCTAAGCAAAATGTTAGC-3' (plus strand primer #584) (SEQ ID NO. 17) ; 5' -GCGTTCAAGTACTCCAGA-3' (minus strand primer #200) (SEQ ID NO. 18) ; 5' -GATATCTAGATCTTATTTTAAAGCGTT-3' (minus strand primer #586) (SEQ ID NO. 19); and 5' -GGATCCGGTGACCTTTTAAAGCGTTTTTAAT-3' (minus strand primer #1169) (SEQ ID NO. 20) . Cloning and Sequencing of OspB Similar methods were also used to isolate OspB genes. One OspB genes isolated is represented as SEQ ID NO. 21 (OspB-B31) ; its encoded amino acid sequence is SEQ ID NO. 22. The following primers were used to generate specific nucleic acid sequences of the OspB gene, to be used in generation of chimeric nucleic acid sequences (see Example 4) : 5'-GGTACAATTACAGTACAA-3' (plus strand primer #721) (SEQ ID NO. 23) ; 5' -CCGAGAATCTCATATGGCACAAAAAGGTGCTGAGTCAATTGG-3' (plus strand primer #1105) (SEQ ID NO. 24) ; 5' -CCGATATCGGATCCTATTTTAAAGCGTTTTTAAGC-3' (minus strand primer # 1106) (SEQ ID NO. 25); and 5' -GGATCCGGTGACCTTTTAAAGCGTTTTTAAG-3' (minus strand primer #1170) (SEQ ID NO. 26) . Cloning and Sequencing of OspC Similar methods were also used to isolate OspC genes. The following primers were used to isolate entire OspC genes from Borrelia strains B31, K48, PKO, and pTrob: 5' -GTGCGCGACCATATGAAAAAGAATACATTAAGTGCG-3' (plus strand primer having Ndel site combined with start codon) (SEQ ID NO. 27) , and 5' -GTCGGCGGATCCTTAAGGTTTTTTTGGACTTTCTGC-3' (minus strand primer having BamHl site followed by stop codon) (SEQ ID NO. 28) . The nucleic acid sequences of the OspC genes were then determined by the dideoxy chain-termination technique using the Sequenase kit (United States Biochemical) . OspC genes isolated and sequenced in this manner include those for strains B31, K48, PKo, and Tro; the nucleic acid sequences are depicted in the sequence listing as SEQ ID NO. 29 (OspC-B31) , SEQ ID NO. 31 (OspC-K48) , SEQ ID NO. 33 (OspC-PKo) , and SEQ ID NO. 35 (OspC-Tro) . An alignment of these sequences is shown in Figure 38. The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO. 30 (OspC-B31) , SEQ ID NO. 32 (OspC-K48), SEQ ID NO. 34 (OspC-PKo) , and SEQ ID NO. 36 (OspC-Tro) . Truncated OspC genes were generated using other primers. These primers were designed to amplify nucleic acid sequences, derived from the OspC gene, that lacked the nucleic acids encoding the signal peptidase sequence of the full-length protein. The primers corresponded to bp 58-75 of the natural protein, with a codon for Met-Ala attached ahead. For strain B31, the following primer was used: 5' -GTGCGCGACCATATGGCTAATAATTCAGGGAAAGAT-3' (SEQ ID NO. 37) . For strain PKo, 5' -GTGCGCGACCATATGGCTAGTAATTCAGGGAAAGGT-3' (SEQ ID NO. 38) was used. For strains pTrob and K48, 5' -GTGCGCGACCATATGGCTAATAATTCAGGTGGGGAT-3' (SEQ ID NO. 39) was used. Additional primers were also designed to amplify nucleic acids encoding particular polypeptides, for use in creation of chimeric nucleic acid sequences (see Example 4) . These primers included: 5' -CTTGGAAAATTATTTGAA-3' (plus strand primer #520) (SEQ ID NO. 40) ; 5'-CACGGTCACCCCATGGGAAATAATTCAGGGAAAGG-3' (plus strand primer #58) (SEQ ID NO. 41) ; 5' -TATAGATGACAGCAACGC-3' (minus strand primer #207) (SEQ ID NO. 42) ; and 5' -CCGGTGACCCCATGGTACCAGGTTTTTTTGGACTTTCTGC-3' (minus strand primer #636) (SEQ ID NO. 43) . Cloning and Sequencing of OspD Similar methods can be used to isolate OspD genes. An alignment of four OspD nucleic acid sequences (from strains pBo, PGau, DK29, and K48) is shown in Figure 39. Cloning and Sequencing of pl2 The pl2 gene was similarly identified. Primers used to clone the entire pl2 gene included: 5'- CCGGATCCATATGGTTAAAAAAATAATATTTATTTC-3' (forward primer # 757) (SEQ ID NO. 44) ; and 5'- GATATCTAGATCTTTAATTGCTCTGCTCACTCTCTTC-3' (reverse primer #758) (SEQ ID NO. 45) . To amplify a truncated pl2 gene (one in which the transcribed protein is non-lipidated, and begins at amino acid 18 of the native sequence) , the following primers were used: 5' -CCGGGATCCATATGGCTAGTGCAATTGGTCGTGG-3' (forward primer # 759) (SEQ ID NO. 46) ; and primer #758 (SEQ ID NO. 45) . Cloning and Sequencing of p41 (fla) A similar approach was used to clone and sequence genes encoding the p41 (fla) protein. The p4l sequences listed in Table II with GenBank accession numbers were isolated using the following primers from strain B31: 5'-ATGATTATCAATCATAAT-3' (+1 to +18) (SEQ ID NO. 47) ; and 5'-TCTGAACAATGACAAAAC-3' (+1008 to +991) (SEQ ID NO. 48) " . The nucleic acid sequences of p41 isolated in this manner are depicted in the sequence listing as SEQ ID NO. 51 (p41- PGau) , and SEQ ID NO. 53 (p41-DK29) . An alignment of several p41 nucleic acid sequences, including those for strains B31, pKal, PGau, pBo, DK29, and pKo, is shown in Figure 41. The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO. 50 (p41-K48) , SEQ ID NO. 52 (p41-PGau) , SEQ ID NO. 54 (p41-DK29) , SEQ ID NO. 56 (p41-PTrob) , and SEQ ID NO. 58 (p41-PHei) . Other primers were designed to amplify nucleic acid sequences encoding polypeptides of p41, to be used in chimeric nucleic acid sequences. These primers included: 5' -TTGGATCCGGTCACCCCATGGCTCAATATAACCAATG-3' (minus strand primer #122) (SEQ ID NO. 59) ; 5' -TTGGATCCGGTCACCCCATGGCTTCTCAAAATGTAAG-3' (plus strand primer # 140) (SEQ ID NO. 60) ; 5' -TTGGATCCGGTGACCAACTCCGCCTTGAGAAGG-3' (minus strand primer # 234) (SEQ ID NO. 61); and 5' -TTGGATCCGGTGACCTATTTGAGCATAAGATGC-3' (minus strand primer #141) (SEQ ID NO. 62) . Cloning and Sequencing of p93 The same approach was also used to clone and sequence p93 protein. Genes encoding p93, as listed in Table II with GenBank accession numbers, were isolated by this method with the following primers from strain B31: 5'-GGTGAATTTAGTTGGTAAGG-3' (-54 to -35) (SEQ ID NO. 63) ; and 5' -CACCAGTTTCTTTAAGCTGCTCCTGC-3' (+1117 to +1092) (SEQ ID NO. 64) . The nucleic acid sequences of p93 isolated in this manner are depicted in the sequence listing as SEQ ID NO. 65 (p93-B31) , SEQ ID NO. 67 (p93-K48) SEQ ID NO. 69 (p93- ' PBo) , SEQ ID NO. 71 (p93-PTrob) , SEQ ID NO. 73 (p93-PGau), SEQ ID NO. 75 (p93-25015), and SEQ ID NO. 77 (p93-PKo) . The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO. 66 (p93-B31) , SEQ ID NO. 68 (p93-K48) SEQ ID NO. 70 (p93-PBo) , SEQ ID NO. 72 (p93-PTrob), SEQ ID NO. 74 (p93-PGau) , SEQ ID NO. 76 (p93-25015) , and SEQ ID NO. 78 (p93-PKo) . Other primers were used to amplify nucleic acid sequences encoding polypeptides of p93 to be used in generating chimeric nucleic acid sequences. These primers included: 5' -CCGGTCACCCCATGGCTGCTTTAAAGTCTTTA-3' (plus strand primer #475) (SEQ ID NO. 79); 5' -CCGGTCACCCCATGAATCTTGATAAAGCTCAG-3' (plus strand primer #900) (SEQ ID NO. 80) ; 5' -CCGGTCACCCCATGGATGAAAAGCTTTTAAAAAGT-3' (plus strand primer #1168) (SEQ ID NO. 81); 5' -CCGGTCACCCCCATGGTTGAGAAATTAGATAAG-3' (plus strand primer #1423) (SEQ ID NO. 82); and 5' -TTGGATCCGGTGACCCTTAACTTTTTTTAAAG-3' (minus strand primer # 2100) (SEQ ID NO. 83) . C. Expression of Proteins from Borrelia Genes The nucleic acid sequences described above can be incorporated into expression plasmids, using standard techniques, and transfected into compatible host cells in order to express the proteins encoded by the nucleic acid sequences. As an example, the expression the pl2 gene and the isolation of pl2 protein is set forth. Amplification of the pl2 nucleic acid sequence was conducted with primers that included a Ndel restriction site into the nucleic acid sequence. The PCR product was extracted with phenol/chloroform and precipitated with ethanol. The precipitated product was digested and ligated into an expression plasmid as follows: 15 μl (approximately 1 μg) of PCR DNA was combined with 2 μl 10X restriction buffer for Ndel (Gibco/BRL) , 1 μl Ndel (Gibco/BRL) , and 2 μl distilled water, and incubated overnight at 37°C. This mixture was subsequently combined with 3 μl 10X buffer (buffer 3, New England BioLabs) , 1 μl BamHI (NEB) , and 6 μl distilled water, and incubated at 37° for two hours. The resultant material was purified by preparative gel electrophoresis using low melting point agarose, and the band was visualized under long wave ultraviolet light and excised from the gel. The gel slice was treated with Gelase using conditions recommended by the manufacturer (Epicentre Technologies) . The resulting DNA pelled was resuspended in 25-50 μl of 10 mM TRIS-CL (pH 8.0) and 1 mM EDTA (TE) . An aliquot of this material was ligated into the Pet9c expression vector (Dunn, J. J. et al . , Protein Expression and Purification 1: 159 (1990)) . To ligate the material into the Pet9c expression vector, 20-50 ng of pl2 nucleic acid sequences cut and purified as described above was combined with 5 μl 10 One- Phor-All (OPA) buffer (Pharmacia) , 30-60 ng Pet9c cut with Ndel and BamHI, 2.5 μl 20 mM ATP, 2 μl T4 DNA ligase (Pharmacia) diluted 1:5 in IX OPA buffer, and sufficient distilled water to bring the final volume to 50 μl. The mixture was incubated at 12°C overnight. The resultant ligations were transformed into competent DH5-alpha cells and plated on nutrient agar plates containing 50 μg/ml kanamycin and incubated overnight at 37 °C. DH5-alpha is used as a "storage strain" for T7 expression clones, because it is RecA deficient, so that recombination and concatenation are not problematic, and because it lacks the T7 RNA polymerase gene necessary to express the cloned gene. The use of this strain allows for cloning of potentially toxic gene products while minimizing the chance of deletion and/or rearrangement of the desired genes. Other cell lines having similar properties may also be used. Kanamycin resistant colonies were single-colony purified on nutrient agar plates supplemented with kanamycin at 50 μg/ml. A colony from each isolate was inoculated into 3-5 ml of liquid medium containing 50 μg/ml kanamycin, and incubated at 37°C without agitation. Plasmid DNA was obtained from 1 ml of each isolate using a hot alkaline lysis procedure (Mantiatis, T. et al . , Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) ) . Plasmid DNA was digested with EcoRI and Bglll in the following manner: 15 μl plasmid DNA was combined with 2 μl 10X buffer 3 (NEB) , 1 μ EcoRI (NEB) , 1 μl Bglll (NEB) and 1 μl distilled water, and incubated for two hours at 37°C. The entire reaction mixture was electrophoresed on an analytical agarose gel. Plasmids carrying the pl2 insert were identified by the presence of a band corresponding to 925 base-pairs (full length pl2) or 875 base-pairs (nonlipidated pl2) . One or two plasmid DNAs from the full length and nonlipidated pl2 clones in Pet9c were used to transform BL21 DE3 pLysS to kanamycin resistance as described by Studier et al . (Methods in Enzymologv, Goeddel, D. (Ed.), Academic Press, 185: 60-89 (1990)) . One or two transformants of the full length and nonlipidated clones were single-colony purified on nutrient plates containing 25 μg/ml chloramphenicol (to maintain pLysS) and 50 μg/ml kanamycin at 37 °C. One colony of each isolate was inoculated into liquid medium supplemented with chloramphenicol and kanamycin and incubated overnight at 37°C. The overnight culture was subcultured the following morning into 500 ml of liquid broth with chloramphenicol (25 μg/ml) and kanamycin (50 μg/ml) and grown with aeration at 37°C in an orbital air-shaker until the absorbance at 600 nm reached 0.4-0.7. Isopropyl-thio-galactoside (IPTG) was added to a final concentration of 0.5 mM, for induction, and the culture was incubated for 3-4 hours at 37° as before. The induced cells were pelleted by centrifuga ion and resuspended in 25 ml of 20 mM NaP0 4 (pH 7.7) . A small aliquot was removed for analysis by gel electrophoresis. Expressing clones produced proteins which migrated at the 12 kDa position. A crude cell lysate was prepared from the culture as described for recombinant OspA by Dunn, J.J. et al . , (Protein Expression and Purification 1: 159 (1990) ) . The crude lysate was first passed over a Q-sepharose column (Pharmacia) which had been pre-equilibrated in Buffer A: 10 mM NaP0 4 (pH 7.7), 10 mM NaCl, 0.5 mM PMSF. The column was washed with 10 mM NaP0 4 , 50 mM NaCl and 0.5 mM PMSF and then pl2 was eluted in 10 mM NaP0 4 , 0.5 mM PMSF with a NaCl gradient from 50-400 mM. pl2 eluted approximately halfway through the gradient between 100 and 200 mM NaCl. The peak fractions were pooled and dialyzed against 10 mM NaPo4 (pH 7.7) , 10 mM NaCl, 0.5 mM PMSF. The protein was then concentrated and applied to a Sephadex G50 gel filtration column of approximately 50 ml bed volume (Pharmacia) , in 10 mM NaP0 4 , 200 mM NaCl, 0.5 mM PMSF. pl2 would typically elute shortly after the excluded volume marker. Peak fractions were determined by running small aliquots of all fractions on a gel. The pl2 peak was pooled and stored in small aliquots at -20°C. Example 4. Generation of Chimeric Nucleic Acid Sequences and Chimeric Proteins A. General Protocol for Creation of Chimeric Nucleic Acid Sequences The megaprimer method of site directed mutagenesis and its modification were used to generate chimeric nucleic acid sequences (Sarkar and Som er, Biotechniσues 8(4) : 404- 407 (1990) ; Aiyar, A. and J. Leis, Biotechniσues 14(3) : 366-369 (1993)) . A 5' primer for the first genomic template and a 3' fusion oligo are used to amplify the desired region, the fusion primer consists of a 3' end of the first template (DNA that encodes the amino-proximal polypeptide of the fusion protein) , coupled to a 5' end of the second template (DNA that encodes the carboxy-proximal polypeptide of the fusion protein) . The PCR amplifications are performed using Taq DNA polymerase, 10X PCR buffer, and MgCl 2 (Promega Corp., Madison, WI) , and Ultrapure dNTPs (Pharmacia, Piscataway, NJ) . One μg of genomic template 1, 5 μ of 10 μM 5' oligo and 5 μl of 10 μM fusion oligo are combined with the following reagents at indicated final concentrations: 10X Buffer-Mg FREE (IX) , MgCl 2 (2 mM) , dNTP mix (200 μM each dNTP) , Taq DNA polymerase (2.5 units) , water to bring final volume to 100 μl. A Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) is used to amplify under the following conditions: 35 cycles at 95°C for one minute, 55°C for two minutes, and 72° for three minutes. This procedure results in a "megaprimer" . The resulting megaprimer is run on a IX TAE, 4% low- melt agarose gel. The megaprimer band is cut from the gel and purified using the Promega Magic PCR Preps DNA purification system. Purified megaprimer is then used in a second PCR step. One μg of genomic template 2, approximately 0.5 μg of the megaprimer, and 5 μ of 10 μM 3' oligo are added to a cocktail of 10X buffer, MgCl 2 , dNTPs and Taq at the same final concentrations as noted above, and brought to 100 μl with water. PCR conditions are the same as above. The fusion product resulting from this amplification is also purified using the Promega Magic PCR Preps DNA purification system. The fusion product is then ligated into TA vector and transformed into E. coli using the Invitrogen (San Diego, CA) TA Cloning Kit. Approximately 50 ng of PCR fusion product is ligated to 50 ng of pCRII vector with IX Ligation Buffer, 4 units of T4 ligase, and brought to 10 Nl with water. This ligated product mixture is incubated at 12°C overnight (approximately 14 hours) . Two μl of the ligation product mixture is added to 50 μl competent INC F' cells and 2 μ beta mercaptoethanol. The cells are then incubated for 30 minutes, followed by heat shock treatment at 42°C for 60 seconds, and an ice quenching for two minutes. 450 μl of warmed SOC media is then added to the cells, resulting in a transformed cell culture which is incubated at 37°C for one hour with slight shaking. 50 μl of the transformed cell culture is plated on LB + 50 μg/μl ampicillin plates and incubated overnight at 37°C. Single white colonies are picked and added to individual overnight cultures containing 3 ml LB with ampicillin (50 μg/μl) . The individual overnight cultures are prepared using Promega's Magic Miniprep DNA purification system. A small amount of the resulting DNA is cut using a restriction digest as a check. DNA sequencing is then performed to check the sequence of the fusion nucleic acid sequence, using the United States Biochemical (Cleveland, OH) Sequenase Version 2.0 DNA sequencing kit. Three to five μg of plasmid DNA is used per reaction. 2 μl 2M NaOH/2mM EDTA are added to the DNA, and the volume is brought to 20 μl with water. The mixture is then incubated at room temperature for five minutes. 7 μl water, 3μl 3M NaAc, 75 μl EtOH are added. The resultant mixture is mixed by vortex and incubated for ten minutes at -70°C, and then subjected to microfugation. After microfuge for ten minutes, the supernatant is aspirated off, and the pellet is dried in the speed vac for 30 second. 6 μl water, 2 μl annealing buffer, and 2 μl of 10 μM of the appropriate oligo is then added. This mixture is incubated for 10 minutes at 37°C and then allowed to stand at room temperature for 10 minutes. Subsequently, 5.5 μl of label cocktail (described above) is added to each sample of the mixture, which are incubated at room temperature for an additional five minutes. 3.5 μl labeled DNA is then added to each sample which is then incubated for five minutes at 37°C. 4 μl stop solution is added to each well. The DNA is denatured at 95° for two minutes, and then placed on ice. Clones with the desired fusion nucleic acid sequences are then recloned in frame in the pEt expression system in the lipidated (full length) and non-lipidated (truncated, i.e., without first 17 amino acids) forms. The product is amplified using restriction sites contained in the PCR primers. The vector and product are cut with the same enzymes and ligated together with T4 ligase. The resultant plasmid is transformed into competent E. coli using standard transformation techniques. Colonies are screened as described earlier and positive clones are transformed into expression cells, such as E. coli BL21, for protein expression with IPTG for induction. The expressed protein in its bacterial culture lysate form and/or purified form is then injected in mice for antibody production. The mice are bled, and the sera collected for agglutination, in vi tro growth inhibition, and complement- dependent and - independent lysis tests. B. Specific Chimeric Nucleic Acid Sequences Various chimeric nucleic acid sequences were generated. The nucleic acid sequences are described as encoding polypeptides from Borrelia proteins. The chimeric nucleic acid sequences are produced such that the nucleic acid sequence encoding one polypeptide is in the same reading frame as the nucleic acid sequence encoding the next polypeptide in the chimeric protein sequence encoded by the chimeric nucleic acid sequence. The proteins are listed sequentially (in order of presence of the encoding sequence) in the description of the chimeric nucleic acid sequence. For example, if a chimeric nucleic acid sequence consists of bp 1-650 from OspA-1 and bp 651-820 from OspA-2 were sequenced, the sequence of the chimer would include the first 650 base pairs from OspA-1 followed immediately by base pairs 651-820 of OspA-2. OspA-K48/OspA-PGau A chimer of OspA from strain K48 (OspA-K48) and OspA from strain PGau (OspA-PGau) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-654 from OspA-K48, followed by bp 655-820 from OspA-PGau. Primers used included: the amino-terminal sequence of OspA primer #607 (SEQ ID NO. 16) ; the fusion primer, 5' -AAAGTAGAAGTTTTTGAATCCCATTTTCCAGTTTTTTT-3' (minus strand primer #668-654) (SEQ ID NO. 84) ; the carboxy-terminal sequence of OspA primer #586 (SEQ ID NO. 19) ; and the sequence primers #369 (SEQ ID NO. 14) and #357 (SEQ ID NO. 15) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 85; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 86. OspA-B31/OspA-PGau A chimer of OspA from strain B31 (OspA- B31) and OspA from strain PGau (OspA-PGau) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-651 from 0spA-B31, followed by bp 652-820 from OspA-PGau. Primers used included: the fusion primer, 5'-AAAGTAGAAGTTTTTGAATTCCAAGCTGCAGTTTT-3' (minus strand primer #668-651) (SEQ ID NO. 87) ; and the sequence primer, #369 (SEQ ID NO. 14) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 88; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 89. OSPA-B31/OspA-K48 A chimer of OspA from strain B31 (OspA- B31) and OspA from strain K48 (OspA-K48) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-651 from OspA-B31, followed by bp 652-820 from OspA-K48. Primers used included: the fusion primer, 5' -AAAGTGGAAGTTTTTGAATTCCAAGCTGCAGTTTTTTT-3' (minus strand primer #671-651) (SEQ ID NO. 90) ; and the sequence primer, #369 (SEQ ID NO. 14) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 91; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 92. OspA-B31/OspA-25015 A chimer of OspA from strain B31 (OspA- B31) and OspA from strain 25015 (OspA-25015) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-651 from OspA-B31, followed by bp 652-820 from OspA-25015. Primers used included: the fusion primer, 5' -TAAAGTTGAAGTGCCTGCATTCCAAGCTGCAGTTT-3' (SEQ ID NO. 93) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 94; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 95. OspA-K48/OspA-B31/OspA-K 8 A chimer of OspA from strain B31 (0spA-B31) and OspA from strain K48 (OspA-K48) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-570 from OspA-B31, followed by bp 570-651 from OspA-B31, followed by bp 650- 820 from OspA-K48. Primers used included: the fusion primer, 5' -CCCCAGATTTTGAAATCTTGCTTAAAACAAC-3' (SEQ ID NO. ' 96) ; and the sequence primer, #357 (SEQ ID NO. 15) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 97; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 98. OspA-B31/OspA-K48/OspA-B31/OspA-K48 A chimer of OspA from strain B31 (OspA-B31) and OspA from strain K48 (OspA- K48) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-420 from OspA- B31, followed by 420-570 from OspA-K48, followed by bp 570- 650 from OspA-B31, followed by bp 651-820 from OspA-K48. Primers used included: the fusion primer, 5'- CAAGTCTGGTTCCAATTTGCTCTTGTTATTAT-3' (minus strand primer #436-420) (SEQ ID NO. 99); and the sequence primer, #357 (SEQ ID NO. 15) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 100; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 101. OSPA-B31/QSPB-B31 A chimer of OspA and OspB from strain B31 (OspA-B31, OspB-B31) was generated using the method described above. The chimeric nucleic acid sequence included bp 1-651 from OspA-B31, followed by bp 652-820 from OspB-B31. Primers used included: the fusion primer, 5' -GTTAAAGTGCTAGTACTGTCATTCCAAGCTGCAGTTTTTTT-3' (minus strand primer #740-651) (SEQ ID NO. 102) ; the carboxy- terminal sequence of OspB primer #1106 (SEQ ID NO. 25) ; and the sequence primer #357 (SEQ ID NO. 15) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 103; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 104. OspA-B31/OspB-B3l/OspC-B31 A chimer of OspA, OspB and OspC from strain B31 (OspA-B31, OspB-B31, and OspC-B31) was generated using the method described above. The chimeric nucleic acid sequence included bp 1-650 from OspA-B31, followed by bp 652-820 from OspB-B31, followed by bp 74-630 of OspC-B31. Primers used included: the fusion primer, 5'- TGCAGATGTAATCCCATCCGCCATTTTTAAAGCGTTTTT-3' (SEQ ID NO. 105) ; and the carboxy-terminal sequence of OspC primer (SEQ ID NO. 28) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 106; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 107. OspC-B3l/OspA-B31/OspB-B31 A chimer of OspA, OspB and OspC from strain B31 (OspA-B31, OspB-B31, and OspC-B31) was generated using the method described above. The chimeric nucleic acid sequence included bp 1-630 from OspC-B31, followed by bp 52-650 from OspA-B31, followed by bp 650-820 of OspB-B31. Primers used included: the amino-terminal sequence of OspC primer having SEQ ID NO. 27; the fusion primer, 5' -GCTGCTAACATTTTGCTTAGGTTTTTTTGGACTTTC-3' (minus strand primer #69-630) (SEQ ID NO. 108) ; and the sequence primers #520 (SEQ ID NO. 40) and #200 (SEQ ID NO. 18) . The chimeric nucleic acid sequence is presented as SEQ ID NO. 109; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO. 110. Additional Chimeric Nucleic Acid Sequences Using the methods described above, other chimeric nucleic acid sequences were produced. These chimeric nucleic acid sequences, and the proteins encoded, are summarized in Table 3. Table III Chimeric Nucleic acid Sequences and the Encoded Proteins C. Purification of Proteins Generated by Chimeric Nucleic Acid Sequences The chimeric nucleic acid sequences described above, as well as chimeric nucleic acid sequences produced by the methods described above, are used to produce chimeric proteins encoded by the nucleic acid sequences. Standard methods, such as those described above in Example 3, concerning the expression of proteins from Borrelia genes, can be used to express the proteins in a compatible host organism. The chimeric proteins can then be isolated and purified using standard techniques. If the chimeric protein is soluble, it can be purified on a Sepharose column. Insoluble proteins can be solubilized in guanidine and purified on a Ni++ column; alternatively, they can be solubilized in 10 mM NaP0 4 with 0.1 - 1% TRIXON X 114, and subsequently purified over an S column (Pharmacia) . Lipidated proteins were generally purified by the latter method. Solubility was determined by separating both soluble and insoluble fractions of cell lysate on a 12% PAGE gel, and checking for the localization of the protein by Coomasie staining, or by Western blotting with monoclonal antibodies directed to an antigenic polypeptide of the chimeric protein. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. such equivalents are intended to be encompassed in the scope of the following claims.

Claims: CLAIMS What is claimed is: 1. A chimeric protein comprising two or more antigenic Borrelia polypeptides, wherein the antigenic Borrelia polypeptides which comprise the chimeric protein do not occur naturally in the same protein in Borrelia. 2. The chimeric protein of Claim 1, wherein the antigenic Borrelia polypeptides are from two or more different species of Borrelia . 3. The chimeric protein of Claim 2, wherein the antigenic Borrelia polypeptides are derived from Borrelia proteins selected from the group consisting of: outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 4. The chimeric protein of Claim 3, wherein the antigenic Borrelia polypeptides are from corresponding proteins from two or more different species of Borrelia . 5. The chimeric protein of Claim 3, wherein the antigenic Borrelia polypeptides are from non-corresponding proteins from at least two different species of Borrelia. 6. The chimeric protein of Claim 1, wherein two or more antigenic Borrelia polypeptides are from the same species of Borrelia. 7. The chimeric protein of Claim 6, wherein the antigenic Borrelia polypeptides are derived from Borrelia proteins selected from the group consisting of: outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 8. The chimeric protein of Claim 7, wherein the antigenic Borrelia polypeptides are from the same protein. 9. The chimeric protein of Claim 6, wherein the antigenic Borrelia polypeptides are from different proteins. 10. A chimeric protein comprising two antigenic Borrelia polypeptides flanking a tryptophan residue, wherein the amino-proximal polypeptide consists of a polypeptide that is proximal from the single tryptophan residue of a first outer surface protein of Borrelia, and the carboxy-proximal polypeptide consists of a polypeptide that is distal from the single tryptophan residue of a second outer surface protein of Borrelia . 11. The chimeric protein of Claim 10, wherein the first and second outer surface proteins are from the same species of Borrelia . 12. The chimeric protein of Claim 11, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 13. The chimeric protein of Claim 11, wherein the first outer surface protein is outer surface protein B, and the second outer surface protein is outer surface protein A. 14. The chimeric protein of Claim 10, wherein the first and second outer surface proteins are from different species of Borrelia . 15. The chimeric protein of Claim 14, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 16. The chimeric protein of Claim 14, wherein the first outer surface protein is outer surface protein B, and the second outer surface protein is outer surface protein A. 17. The chimeric protein of Claim 14, wherein the first and second outer surface proteins are corresponding proteins selected from the group consisting of: outer surface protein A and outer surface protein B. 18. The chimeric protein of Claim 10, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 19. The chimeric protein of Claim 18, wherein the amino- proximal polypeptide further comprises a first, second, and third hypervariable domain, the first hypervariable domain consisting of residues 120 through 140 of outer surface protein A, the second hypervariable domain consisting of residues 150 through 180 of outer surface protein A, and the third hypervariable domain consisting of residues 200 through 217 of outer surface protein A. 20. The chimeric protein of Claim 19, wherein the first and second hypervariable domains are derived from outer surface protein A from different species of Borrelia. 21. The chimeric protein of Claim 10, further comprising an antigenic Borrelia polypeptide derived from a Borrelia protein selected from the group consisting of: outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 22. A nucleic acid sequence encoding a chimeric protein comprising two antigenic Borrelia polypeptides, wherein the two antigenic Borrelia polypeptides which comprise the chimeric protein do not occur naturally in the same protein in Borrelia . 23. The nucleic acid sequence of Claim 22, wherein the antigenic Borrelia polypeptides are from two or more different species of Borrelia . 24. The nucleic acid sequence of Claim 23, wherein the antigenic Borrelia polypeptides are derived from Borrelia proteins selected from the group consisting of: outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 25. The nucleic acid sequence of Claim 24, wherein the antigenic Borrelia polypeptides are from corresponding proteins from two or more different species of Borrelia. 26. The nucleic acid sequence of Claim 24, wherein two or more of the antigenic Borrelia polypeptides are from non-corresponding proteins from different species of Borrelia . 27. The nucleic acid sequence of Claim 22, wherein two or more antigenic Borrelia polypeptides are from the same species of Borrelia. 28. The nucleic acid sequence of Claim 27, wherein the antigenic Borrelia polypeptides are derived from Borrelia proteins selected from the group consisting of: outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 29. The nucleic acid sequence of Claim 28, wherein the antigenic Borrelia polypeptides are from the same protein. 30. The nucleic acid sequence of Claim 27, wherein the antigenic Borrelia polypeptides are from different proteins. 31. A nucleic acid sequence encoding a chimeric protein comprising two antigenic Borrelia polypeptides flanking a tryptophan residue, wherein the amino- proximal polypeptide consists of a polypeptide that is proximal from the single tryptophan residue of a first outer surface protein of Borrelia, and the carboxy- proximal polypeptide consists of a polypeptide that is distal from the single tryptophan residue of a second outer surface protein of Borrelia . 32. The nucleic acid sequence of Claim 31, wherein the first and second outer surface proteins are from the same species of Borrelia . 33. The nucleic acid sequence of Claim 32, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 34. The nucleic acid sequence of Claim 32, wherein the first outer surface protein is outer surface protein B, and the second outer surface protein is outer surface protein A. 35. The nucleic acid sequence of Claim 31, wherein the first and second outer surface proteins are from different species of Borrelia . 36. The nucleic acid sequence of Claim 35, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 37. The nucleic acid sequence of Claim 35, wherein the first outer surface protein is outer surface protein B, and the second outer surface protein is outer surface protein A. 38. The nucleic acid sequence of Claim 35, wherein the first and second outer surface proteins are corresponding proteins selected from the group consisting of: outer surface protein A and outer surface protein B. 39. The nucleic acid sequence of Claim 31, wherein the first outer surface protein is outer surface protein A and the second outer surface protein is outer surface protein B. 40. The nucleic acid sequence of Claim 39, wherein the amino-proximal polypeptide further comprises a first and a second hypervariable domain, the first hypervariable domain consisting of amino acid residues 1 through 140 of outer surface protein A, and the second hypervariable domain consisting of amino acid residues 150 through 217 of outer surface protein A. 41. The nucleic acid sequence of Claim 40, wherein the first and second hypervariable domains are derived from outer surface protein A from different species of Borrelia . 42. The nucleic acid sequence of Claim 31, further comprising an antigenic Borrelia polypeptide derived from a Borrelia protein selected from the group consisting of : outer surface protein A, outer surface protein B, outer surface protein C, outer surface protein D, pl2, p39, p41, p66, and p93. 43. A nucleic acid sequence having a sequence selected from the group consisting of: SEQ ID NO. 85, SEQ ID NO. 88, SEQ ID NO. 91, SEQ ID NO. 94, SEQ ID NO. 97, SEQ ID NO. 100, SEQ ID NO. 103, SEQ ID NO. 106, SEQ ID NO. 109, SEQ ID NO. Ill, SEQ ID NO. 113, SEQ ID NO. 115, SEQ ID NO. 117, SEQ ID NO. 119, SEQ ID NO. 121, . SEQ ID NO. 137, SEQ ID NO. 139, SEQ ID NO. 141, and SEQ ID NO. 143. 44. A protein having an amino acid sequence selected from the group consisting of: SEQ ID NO. 86, SEQ ID NO. 89, SEQ ID NO. 92, SEQ ID NO. 95, SEQ ID NO. 98, SEQ ID NO. 101, SEQ ID NO. 104, SEQ ID NO. 107, SEQ ID NO. 110, SEQ ID NO. 112, SEQ ID NO. 114, SEQ ID NO. 116, SEQ ID NO. 118, SEQ ID NO. 120, SEQ ID NO. 122, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, and SEQ ID NO. 144. 45. A chimeric protein according to any one of claims 1 to 21 and 44 for use in therapy or diagnosis, for example as a vaccine against Borrelia infection, in immunodiagnostic assays to detect the presence of antibodies to Borrelia or to measure T-cell reactivity. 46. A chimeric protein according to claim 45, wherein the immunodiagnostic assay is a dot blot, Western blot, . ELISA or agglutination assay. 47. Use of the chimeric protein according to any one of claims 1 to 21 and 44, or the nucleic acid sequence of any one of claims 22 to 43, for the manufacture of a compound for use in therapy or diagnosis, for example as a vaccine against Borrelia infection, in immunodiagnostic assays to detect the presence of antibodies to Borrelia or to measure T-cell reactivity. 48. Use according to claim 47, wherein the immunodiagnostic assay is a dot blot, Western blot, ELISA or agglutination assay.

Inventors: DUNN JOHN J, LUFT BENJAMIN J, DUNN, JOHN, J., LUFT, BENJAMIN, J.
Applicants: ASS UNIVERSITIES INC, ASSOCIATED UNIVERSITIES, INC.
Application year: 1994
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N15